A recent general survey of the literature by the PI showed that >1000 articles had been published in 2011-2012 involving some type of cellular assay using microfluidic technologies. Because cellular function in vivo is most often affected by other cell types and tissues, a recent trend in microfluidic cellular assays has been to investigat "organs" or other tissue types on a single device. Such devices will enable cell to cell communication to be monitored on a controlled, in vitro platform. The PI's group has been performing such studies with multiple cell types for nearly a decade now and knows first-hand the complexity of preparing such a device;for example, cell immobilization and culture, introduction and flow of reagents (stimuli, inhibitors, etc.), and detection must all work simultaneously on a single device for success. If one fails, the entire device must be scrapped and began anew. In this application, the PI will build upon recent work by his own group, and others, to create a modular microfluidic system whereby areas for sample (cell) injection, cell perturbation (here red blood cell exposure to regions of varying oxygen concentration), interaction with other cell types, and detection will all be performed on separate components, or modules, that are reversibly sealed to each other. Importantly, if multiple modules are available (e.g., multiple cell growth modules growing in an incubator), failure by one module doesn't mean the experiment is over;that failed module can simply be exchanged for another module and the experiment moves to completion in a timely manner. The PI will demonstrate device utility by investigating an important biological problem currently facing the transfusion medicine community. Specifically, when patients receive a transfusion of red blood cells, there is a corresponding lack of nitric oxide produced in vivo;thus, blood flow is compromised. It is currently suspected by clinical researchers in the blood banking community that the stored red blood cell is the culprit of these reduced nitric oxide levels. The PI and his group will utilize te proposed modular device to demonstrate that the red blood cell does indeed play a role in nitric oxide availability by evaluating red cell response to varying levels of reduced oxygen concentrations (hypoxia). We will also demonstrate that one possible reason for this response is the current solutions being used by the blood banking community to collect and store blood collected from donors. In summary, the PI believes the current storage solutions are rendering the stored red cells non-responsive to hypoxic conditions in the transfusion patient. This reduced response to oxygen tensions is resulting in a decrease in nitric oxide and an overall reduction in blood flow and increase in other post-transfusion complications. The development of the microfluidic device proposed here will facilitate confirmation of this hypothesis about nitrc oxide availability in transfusion medicine. Thus, results from this proposal will be high impact both technologically and from a health-significance platform.

Public Health Relevance

Over the past decade, microfluidic devices have been applied to the analysis of important biomolecules and cells. Now, developers of these devices are being challenged to fabricate devices that incorporate multiple cell types and enable monitoring of cell to cell communication and cellular function under various pharmacological and environmental stimuli. In this application, a modular approach to constructing exactly such a device is described for determining cellular response, and downstream intercellular communication, for stored red blood cells that are used in transfusion medicine.